Hepatitis C is one of the most widespread infectious diseases in the world. About 180 million people are infected with hepatitis C virus (HCV) worldwide with a yearly incidence of 3-4 million. While the acute phase of infection is mostly asymptomatic, the majority of acutely infected individuals develops chronic hepatitis and is at increased risk of developing liver cirrhosis and hepatocellular carcinoma. Thus, HCV infection is a major contributor to end-stage liver disease, and in developed countries to liver transplantation.
HCV is a small, enveloped virus classified as a member of the Flaviviridae family. Its genome consists of a 9.6 kb single stranded RNA of positive polarity composed of 5′ and 3 untranslated regions (UTR) and one long open reading frame (ORF) encoding a polyprotein, which is co- and post-translationally cleaved and thus yields the structural (Core, E1, E2), p7 and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins (Gottwein et al. 2008).
HCV isolates from around the world exhibit significant genetic heterogeneity. At least 7 major HCV genotypes (genotypes 1-7) have been identified, which differ by 31-33% at the nucleotide level and deduced amino acid level. In addition, there are numerous subtypes (a, b, c, etc.), which differ by 20-25% on the nucleotide and deduced amino acid level (Gottwein et al. 2008).
While HCV genotypes 1-3 predominate in the Western World, genotypes 4-6 are more common in areas with high prevalence or even endemic levels of HCV infection. Genotype 6 is highly prevalent in Southeast Asia. Recently, a genotype 7a was discovered in Canadian and Belgian patients, who presumably were infected in Central Africa (Murphy et al., 2007).
While the only approved treatment for chronic HCV infection, combination therapy with interferon-α and ribavirin, leads to a sustained virologic response in most of genotype 2 or 3 patients, viral clearance is only obtained for about half of patients with genotype 1 or 4. There is no vaccine against HCV.
Since its discovery in 1989, research on HCV has been hampered by the lack of appropriate cell culture systems allowing for research on the complete viral life cycle as well as new therapeutics and vaccines.
In 2001, a genotype 2a isolate (JFH1) was described (Kato et al., 2001), which yielded high RNA titers in the replicon system without adaptive mutations (Kato et al., 2003).
A major breakthrough occurred in 2005, when formation of infectious viral particles was reported after transfection of RNA transcripts from the JFH1 full-length consensus cDNA clone into Huh7 cells (Wakita et al., 2005).
At the same time, Lindenbach et al. demonstrated that the intragenotypic 2a/2a recombinant genome (J6/JFH1), in which the structural genes (Core, E1, E2), p7 and NS2 of JFH1 were replaced by the respective genes of clone J6CF, produced infectious viral particles in Huh7.5 cells (a cell line derived from bulk Huh7 cells) with an accelerated kinetic (Lindenbach et al., 2005). Cell culture derived J6/JFH viruses were apparently fully viable in vivo.
Despite the importance of the described cell culture systems they represent only a single subtype (genotype 2a) of HCV. It is important to develop cell culture systems for representative strains of other HCV genotypes, since neutralizing antibodies are not expected to cross-neutralize all genotypes and new specific antiviral compounds might have differential efficiencies against different genotypes.
Thus, the present inventors recently developed JFH1-based intergenotypic recombinants with the structural genes (Core, E1 and E2), p7 and NS2 of all seven major genotypes, depending on the JFH1 genes NS3, NS4A, NS4B, NS5A and NS5B and the JFH1 untranslated regions for efficient replication. These systems allow for genotype-specific functional studies of HCV entry, assembly/release and of the NS2 protease. In addition, these systems can be used for studies of neutralizing antibodies, receptor usage etc. Thus the present inventors previously developed JFH-1 based intergenotypic recombinant viruses of all seven major genotypes.
The HCV NS5A protein plays a vital but undefined role in viral RNA replication. It is a large (56 to 58 kDa) hydrophilic phosphoprotein, divided into three putative principal domains (I-III). The domains are separated by repetitive low-complexity sequences (LCSI and II). The crystal structure of the well-conserved domain I was determined. N-terminal of domain I is located an amphipathic alpha-helix that anchors the protein to membrane structures. Disruption of the amphipathic alpha-helix attenuates replication in the replicon system. Four essential cysteine residues within domain I collectively bind to a single structural zinc ion, and in the replicon system mutation of these residues results in attenuation of RNA replication. The zinc-binding domain exhibit conserved external surfaces, which presumably interact with viral and cellular proteins. Domain I also contains a highly basic channel, which could serve as an RNA-binding pocket during replication. NS5A domains II and III are far less conserved among HCV genotypes than domain I, and its functions are less well understood. Large parts of domain II and III were shown to be disposable for replication in replicon systems. Instead it was suggested that domain III plays an important role during particle formation. Replication efficiency was shown to be influenced by NS5A phosphorylation state. The hyperphosphorylated form (58 kDa) induced by the action of cellular kinases seemed to reduce replication while the basely phosphorylated form (56 kDa) enhanced replication (Gottwein et al. 2008).
A number of studies suggested NS5A to contribute to differential response rates to interferon-alpha in patients infected with genotype 1 versus genotype 2 and 3. In addition, some studies suggested that the sequence of the putative interferon-sensitivity determining region (ISDR) in NS5A domain II has an influence on treatment outcome. Also, binding of NS5A to proteins such as PKR and 2′,5′-oligoadenylate synthetase (2,5-OAS) involved in innate immunity was suggested to be of importance for viral viability (Gottwein et al. 2008).